Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes. These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. Most membrane transport proteins that have been studied in detail have been found to be transmembrane proteins. These proteins enable the specific molecules to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer of the plasma membrane.
There are two major classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins bind the specific molecule to be transported and undergo a series of conformational changes in order to transfer the bound molecule across the membrane. Channel proteins, on the other hand, form hydrophilic pores that extend across the lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Transport through channel proteins occurs at a much faster rate than transport mediated by carrier proteins.
Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the membrane are referred to as voltage gated ion channels (or voltage-dependent ion channels).
Voltage-dependent ion channels are a class of channel proteins that play a major role in cellular electrical excitability. In the majority of excitable tissues, the early depolarization phase of action potentials is mediated by a sodium current via voltage-dependent sodium channels (also known as voltage-gated sodium channels or VGSCs).
The sodium channel is one of the most thoroughly characterized of the voltage gated channels (see FIG. 1 for a model of a voltage sensitive sodium channel). Six distinct neurotoxin or drug receptor sites have been characterized on the sodium channel, associated with channel pore or gating structures (Catterall 1986; Catterall 1988). The mechanisms by which drugs and neurotoxin agonists and antagonists act at the sodium channel and the development of general rules for how drugs interact with other ion channels having extensive homologies to sodium channels can be more readily studied once the actual structure of various sodium channels is more clearly understood.
The primary structures of many sodium channels from a variety of tissues (brain, skeletal muscle and cardiac muscle) and organisms (jellyfish, squid, eel, rat, human) have been identified, and their amino acid sequences show individual regions which are highly conserved over evolution, indicating that voltage-dependent sodium channels belong to a large superfamily of evolutionarily related proteins (Alberts et al. 1994). All published polypeptide complexes of VGSCs have in common a large, about 260 kDa glycoprotein (the pore forming subunit) which is called the alpha subunit (Agnew et al. 1978; Agnew et al. 1980; Catterall 1986; Catterall 1992). Additional lower molecular weight polypeptides, the beta-subunits, have been found to be associated with sodium channels from mammalian muscle (Kraner et al. 1985; Tanaka et al. 1983) and brain (Hartshorne and Catterall 1984). The large, pore-forming alpha subunit is sufficient for all known functions of VGSCs (Catterall 1992; Anderson and Koepe 1992), while the beta subunits modulate some of the functions of the alpha subunit (Catterall 1992; Anderson and Koepe 1992).
Cloning studies of cDNAs encoding the sodium channel large .alpha.-subunit from eel electroplax (Noda et al. 1984), rat brain (Noda et al. 1986b), and Drosophila (Salkoff et al. 1987a; Salkoff et al. 1987b) have demonstrated that: 1) the sequence of the .alpha.-subunit consists of four repeated, highly homologous hydrophobic domains (each of which contains six transmembrane segments of S1-S6) separated by hydrophilic, nonrepeated intervening sequences (see FIG. 2); 2) considerable homology exists among the sequences from different species, with the greatest conservation existing among the four internally homologous domains; 3) the S4 segment of each homologous domain is positively charged, with four to eight lysine or arginine residues at every third position, which may be involved in channel gating (Greenblatt et al. 1985; Guy et al. 1986; Noda et al. 1984); 4) in rat brain (Noda et al. 1986a; Kayano et al. 1988), three homologous genes encode four mRNA sequences (designated as types I, II, IIA, and III) which in turn encode four distinct sodium channel isoforms in the same tissue; and 5) expression of mRNA injected into oocytes, coding for the .alpha.-subunit alone of the rat brain I, II, or III sodium channels, was sufficient to produce a functional voltage-activated sodium channel (Noda et al. 1986a; Suzuki et al. 1988; Agnew 1986; Goldin et al. 1986) exhibiting many of the key properties of the native channel, including appropriate kinetics, voltage-sensitivity, ion selectivity, and sensitivity to the neurotoxin TTX. Different groups have found .beta.-subunits important to varying extents to sodium channel function, making their role somewhat controversial (Catterall 1986; Agnew 1986; Goldin et al. 1986; Messner et al. 1986; Auld et al. 1988; Stuhmer et al. 1987).
The detection of three separate cDNA clones has led to the identification of three structurally distinct sodium channel isoforms in rat brain (Noda et al. 1986a). Two further distinct isoforms have been detected in rat skeletal muscle (Barchi 1988). Another sodium channel isoform was found in rat heart (Rogart 1995).
There continues to exist a need in the art for specific information concerning the primary structural conformation of other sodium channel proteins. Availability of such DNA sequences would make possible the application of recombinant methods to the large scale production of the proteins in prokaryotic and/or eukaryotic host cells, as well as DNA-DNA, DNA-RNA, and RNA-RNA hybridization procedures for the detection, quantification and/or isolation of nucleic acids associated with these proteins. Possession of peripheral nerve sodium channels and related sodium channel proteins and/or knowledge of the amino acid sequences of the same would make possible, in turn, the development of monoclonal and polyclonal antibodies thereto (including antibodies to protein fragments or synthetic peptides modeled thereon) for use in immunological methods for the detection and quantification of the proteins in fluid and tissue samples, as well as for tissue specific delivery of substances such as labels and therapeutic agents to cells expressing the proteins; as well as allowing for the development of new drugs.